Estrogen has the potential to provide potent cardiovascular protection. However, there are mechanisms that modify the vascular actions of estrogen, particularly if disease is already present. In the current funding period we identified 27-hydroxycholesterol (27HC), which is elevated with hypercholesterolemia, as the first endogenous selective estrogen receptor modulator (SERM). We showed that 27HC antagonizes estrogen receptor (ER) function in endothelium and vascular smooth muscle (VSM), and that it causes impaired E2- induced reendothelialization in mice. Recent preliminary studies in mice null for Cyp7b1, which metabolizes 27HC, suggest that 27HC may also promote atherogenesis, attenuate E2-induced protection from neointima formation, and impair E2 regulation of body weight. The latter finding is also likely indicative of impaired glucose homeostasis. Our Overall Objective is to now determine how 27HC contributes directly and indirectly to vascular disease pathogenesis, and how 27HC levels and actions can be modified to lessen vascular disease. Aim 1 is to determine how administered 27HC impacts vascular disease pathogenesis. 27HC-induced changes in atherosclerotic lesion abundance and characteristics will be evaluated in apoE+/+ vs. apoE-/- male and ovariectomized placebo vs. E2-treated female mice. We will also determine how 27HC impacts E2-mediated protection from intimal hyperplasia, and effects of 27HC on E2 regulation of macrophage and VSM cell function will be investigated in culture. Aim 2 is to determine how 27HC impacts body weight regulation and glucose homeostasis, which secondarily influence cardiovascular health. Food intake, energy expenditure and adiposity will be quantified in control vs. 27HC-treated ovariectomized placebo vs. E2-treated female C57BL/6 mice placed on control vs. Western diet. Glucose tolerance and insulin sensitivity and the mechanisms that determine them will be evaluated. CNS vs. peripheral actions of 27HC on ER regulation of metabolism will be distinguished by intraventricular delivery of 27HC. Aim 3 is to determine how prevention of the elevation in 27HC that occurs with hypercholesterolemia impacts the protective actions of E2 on atherosclerosis and metabolism. Serum 27HC will be lowered by hepatic overexpression of Cyp7b1 by IV injection of adenovirus encoding the enzyme and also by creation of a liver-specific Cyp7b1 transgenic mouse. Aim 4 is to develop new strategies to attenuate serum 27HC levels and 27HC actions in target cells. Using a novel extranuclear SERM we developed for in vivo use in mice, the role of non-nuclear ER signaling in hepatic Cyp7b1 upregulation by E2 suggested in cell culture studies will be delineated in vivo. Using peptides identified by phage display to uniquely bind to 27HC-liganded ER, approaches for the selective blockade of 27HC actions mediated by nuclear as well as non-nuclear ER will be pursued in cultured cells. By accomplishing these aims, we will increase our basic understanding of the biology of the only known endogenous SERM in the context of vascular health and disease.